Bacteria are widely used for producing useful substances such as amino acids and proteins. Especially, in recent years, there has been proposed an art in which a useful protein is efficiently produced using a bacterium transformed by genes of a medically- or industrially-useful protein introduced thereinto by genetic engineering techniques.
A bacterium commonly used for protein expression is Escherichia coli that is a gram negative bacterium. The art utilizing Escherichia coli in protein expression has been developed and the art is widely used in production of proteins for industrial, food processing, and medical purposes.
Examples of a method of extracting a protein expressed by using Escherichia coli include physical fracturing methods such as ultrasonication, a method using a high-pressure homogenizer or french press. Such techniques are widely applied in practice. In these physical fracturing methods, Escherichia coli cells are killed when the protein is extracted.
Accordingly, the following methods are effective to increase the amount of the protein to be obtained:
1. Increasing the amount of a recombinant protein expressed per cell; and
2. Increasing the number of cells.
However, the following disadvantages are found in the physical fracturing methods of extracting a recombinant protein from Escherichia coli by breaking Escherichia coli. 
1. Equipment for fracturing Escherichia coli is needed.
2. Nucleic acid constituents such as genomic DNA simultaneously extracted need to be removed from protein extracts for progress of purification, because the nucleic acid constituents may increase a viscosity of the protein extracts.
3. A large amount of proteins derived from Escherichia coli and other impurities contaminated in the protein extracts may cause toxicity and immunogenicity.
4. Purification process is needed in which a large amount of contaminated proteins derived from Escherichia coli is isolated from a recombinant protein.
5. Accumulation of a recombinant protein in Escherichia coli limits the production volume.
6. Inclusion bodies are formed in cells of a bacteria.
7. A required amount of a recombinant protein is not expressed because the recombinant protein is degraded by proteases.
To overcome these disadvantages, a method in which Escherichia coli secretes a recombinant protein in a culture solution is needed.
Extracytoplasmic expression of a recombinant protein by Escherichia coli is commonly carried out by fusing a signal sequence required for inner membrane translocation to a target recombinant protein. Examples of the signal sequence required for inner membrane translocation include signal sequences derived from PelB, OmpA, StII, PhoA, OmpF, PhoE, MalE, OmpC, Lpp, LamB, OmpT, LTB, TorA, and the like, and signal sequences derived from endoxylanase and the like derived from Bacillus. The recombinant protein fused with such a signal sequence is translocated to a periplasm via a Sec pathway or a TAT pathway of an inner membrane translocation mechanism provided in Escherichia coli. However, since a peptidoglycan layer and an outer membrane are provided outside the inner membrane of Escherichia coli, the recombinant protein are commonly not secreted into the culture solution.
Non Patent Documents 1 to 7 each disclose a production method of a recombinant protein by secretion into a culture solution.
The methods disclosed in the above Non Patent Documents each have at least one of the following disadvantages.
1. Since Escherichia coli easily dies, the method does not suit for high-density or continuous production.
2. The method only suits for production of a specific recombinant protein.
3. Since the recombinant protein is expressed as a fusion protein with a membrane protein and the like, the fusion protein may affect the expression of the activity. Accordingly, the process for cutting the fusion protein is needed, which raises costs.
4. The expression amount of the recombinant protein is not adequate.    Non Patent Document 1: Jang et al., Bioproc. Eng., 21 (1999) 453-458    Non Patent Document 2: Yang et al., Appl. Environ. Microbiol., 64 (1998) 2669-2874    Non Patent Document 3: Jeong et al., Appl. Environ. Microbiol., 68 (2002) 4979-4985    Non Patent Document 4: Van der Wal et al., Appl. Environ. Microbiol., 64 (1998) 392-398    Non Patent Document 5: Wan et al., Protein Expr. Purif., 14 (1998) 13-22    Non Patent Document 6: Fernandez et al., Appl. Environ. Microbiol., 66 (2000) 5024-5029    Non Patent Document 7: Li et al., Gene, 25 (2002) 437-447